Understanding the fundamental aspects of autoimmune T cell selection and development is crucial in realizing the breakdown in central tolerance and may be applicable in establishing tolerance to autoimmune TCRs, such as -cell antigen specific T cells. Less than 1% of all thymocytes will recognize self-peptide at high enough affinity/avidity to receive positively selecting survival signals, but also low enough to avoid the activation threshold for apoptosis and deletion. An alternative to negative selection and deletion is the ability of these cells to become unresponsive to antigenic stimulation, i.e. anergy. It is still unclear how the intracellular signaling and genetic signatures differ between T cells selected on naturally expressed high affinity peptides versus low affinity peptides during positive selection. However, even less is known about the signaling events necessary for the selection and survival of autoreactive thymocytes. Only a limited number of TCR transgenic mice specific to islet antigens have been described (BDC2.5, BDC6.9, NY4.1 and 12-4.1), and only one of these TCRs is specific to insulin (12-4.1). This innovative application capitalizes on our strengths and experience in generating TCR retrogenic mice as well as our in depth of knowledge of thymocyte development and TCR signaling. The use of DEC-205 and Clec9a antibody therapy offers a promising avenue to target insulin reactive TCRs as a therapeutic approach. In later studies, we would like to combine other antigens (i.e. chromogranin) with insulin, to target a broader range of TCRs. This combinational therapy may yield better results than individual peptides alone. In addition, we are uniquely positioned to study the development of insulin specific human TCR by utilizing the TCR retrogenic system and the HLA-DQ8 transgenic mouse. Studying the development of T cells expressing TCRs from human INS-VNTR I or III subjects will be extremely informative and innovative. Utilizing humanized mice for the susceptible HLA-DQ8 allele will allow for the first time, studies to be performed detailing the selection and development of autoreative human TCRs. Information gathered from these studies could have a significant impact on our understanding of how autoreactive T cells (both human and mouse) escape negative selection leading to novel approaches targeted towards complete deletion of autoreactive thymocytes and establish central tolerance.